The general objective of this research is to improve our understanding of changes sperm undergo during capacitation by use of ESR spectroscopy and spin label techniques. Using the water soluble spin probe 2H-Tempose, the acrosome volume estimated indirectly by determining the difference between the volume of intact sperm and the volume of sperm from which the acrosome was removed. The average water volume (cubic microns) of sperm with acrosomes (36.6 plus or minus 4.5) was significantly greater than sperm without acrosomes (21.5 plus or minus 3.5) suggesting the acrosome volume was 15.1 cubic microns. For volume determinations of rabbit sperm we observed that the ESR signal from 2H-Tempone decayed after being combined with sperm, which led to inconsistent results. We developed a method which mathematically accounts for this exponential decay and reduces variation in determinations. We also studied the decay reaction rate as a function of sperm concentration and found it to be linear. These data fit a simple model which assumes that enzymes associated with sperm reduce the nitroxide spin probe to the oxime which is not paramanetic. Further studies are required to locate the site of the reaction in the sperm and to determine if the reaction is altered during sperm capacitation. We also determined the water volume of sperm from rabbits ejaculated once daily for four days following 1-2 wk sexual rest and found the average water volume to decline from 15.7 cubic micrometers on the first day to 6.3 cubic microns on the fourth day.